U.S. patent application number 13/055381 was filed with the patent office on 2011-09-01 for treatment of cancers characterized by chromosomal rearrangement of the nut gene.
This patent application is currently assigned to THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Jon Aster, James Bradner, Christopher French, Matthias Hofer.
Application Number | 20110213012 13/055381 |
Document ID | / |
Family ID | 41570839 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110213012 |
Kind Code |
A1 |
French; Christopher ; et
al. |
September 1, 2011 |
Treatment of Cancers Characterized by Chromosomal Rearrangement of
the NUT Gene
Abstract
The present invention is directed, inter alia, to methods of
treating NUT midline carcinoma (NMC) by administering compounds
that promote increased histone acetylation. The invention also
includes assay methods for determining the responsiveness of NMC to
specific histone deacetylases and other compounds.
Inventors: |
French; Christopher;
(Boston, MA) ; Aster; Jon; (Lexington, MA)
; Hofer; Matthias; (Chicago, IL) ; Bradner;
James; (Cambridge, MA) |
Assignee: |
THE BRIGHAM AND WOMEN'S HOSPITAL,
INC.
Boston
MA
DANA-FARBER CANCER INSTITUTE
BOSTON
MA
|
Family ID: |
41570839 |
Appl. No.: |
13/055381 |
Filed: |
July 21, 2009 |
PCT Filed: |
July 21, 2009 |
PCT NO: |
PCT/US09/51328 |
371 Date: |
May 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61129839 |
Jul 23, 2008 |
|
|
|
Current U.S.
Class: |
514/44A ;
514/357; 514/419; 514/431; 514/616 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/15 20130101; A61K 31/4045 20130101; A61K 31/7105 20130101;
A61K 31/4406 20130101; G01N 2800/52 20130101; A61K 31/00 20130101;
G01N 33/574 20130101; A61K 31/343 20130101; A61K 31/165
20130101 |
Class at
Publication: |
514/44.A ;
514/616; 514/431; 514/419; 514/357 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61K 31/16 20060101 A61K031/16; A61K 31/395 20060101
A61K031/395; A61K 31/403 20060101 A61K031/403; A61K 31/4406
20060101 A61K031/4406; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT OF GOVERNMENT FUNDING
[0002] This invention was made with Government support under Grant
No. IR01CA124633-01-5 awarded by the National Institutes of Health.
The U.S. Government therefore has certain rights in the invention.
Claims
1. A method of treating a patient diagnosed as having a cancer with
cells having a NUT chromosomal translocation or where a BRD protein
is present as a translocation partner, said method comprising
administering to said patient a therapeutically effective amount of
a compound that promotes increased acetylation of histones.
2. The method of claim 1, wherein said compound is a histone
deacetylase (HDAC) inhibitor.
3. The method of claim 2, wherein said HDAC inhibitor is selected
from the group consisting of: suberoyl anilide hydroxamic acid
(SAHA); CRA-024781; romidepsin (FK-228); LBH-589 and MS275.
4. (canceled)
5. The method of claim 2, wherein said HDAC inhibitor is
administered to said patient at a daily dose of 10-1000 mg.
6. The method of claim 2, wherein said HDAC inhibitor is
administered to said patient at a daily dose of 50-600 mg.
7. The method of claim 1, wherein said compound is a small
interfering RNA that blocks expression of &aid a NUT-fusion
protein.
8-10. (canceled)
11. The method of claim 1, wherein said cancer is a carcinoma of
the aerodigestive tract or mediastinum of said patient.
12. The method of claim 1, wherein said cancer is a carcinoma of
the nasopharynx; orbit; trachea; pharynx; thymus; posterior
mediastinum; nasal cavity; thorax; sinuses; larynx; or bronchi.
13. The method of claim 1, wherein said cancer is a carcinoma of
the bladder or salivary gland.
14. A method of treating a patient for a solid tumor comprising: a)
assaying cells derived from said solid tumor to determine whether
said cells carry a NUT or BRD chromosomal rearrangement; b) if the
assay of paragraph a) indicates that said chromosomal rearrangement
is present, administering to said patient a therapeutically
effective amount of a compound that promotes increased acetylation
of histones.
15. The method of claim 14, wherein the assay to determine whether
said cells carry a NUT chromosomal rearrangement is a fluorescence
in situ hybridization (FISH) assay.
16. The method of claim 14, wherein the assay to determine whether
said cells carry a NUT chromosomal rearrangement is a reverse
transcriptase polymerase chain reaction assay.
17. The method of claim 14, wherein the assay to determine whether
said cells carry a NUT chromosomal rearrangement is an
immunohistochemical assay to detect expression of NUT or NUT-fusion
protein.
18. The method of claim 14, wherein said compound is a histone
deacetylase (HDAC) inhibitor.
19. The method of claim 18, wherein said HDAC inhibitor is selected
from the group consisting of: SAHA, CRA-024781; trichostatin A,
FK-228, LBH-589 and MS275.
20. (canceled)
21. The method of claim 18, wherein said HDAC inhibitor is
administered to said patient at a daily dose of 10-1000 mg.
22. The method of claim 18, wherein said HDAC inhibitor is
administered to said patient at a daily dose of 50-600 mg.
23. The method of claim 14, wherein said compound is a small
interfering RNA that blocks expression of a NUT-fusion protein.
24-26. (canceled)
27. The method of claim 14, wherein said solid tumor is a carcinoma
of the aerodigestive tract or mediastinum of said patient.
28. The method of claim 27, wherein said solid tumor is a carcinoma
of the nasopharynx; orbit; trachea; pharynx; thymus; posterior
mediastinum; nasal cavity; thorax; sinuses; larynx; salivary gland;
or bronchi.
29-36. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
provisional application 61/129,839, filed on Jul. 23, 2008, the
contents of which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention is concerned with methods of treating
patients with cancers in which there has been a chromosomal
rearrangement that results in a fusion between the NUT (nuclear
protein in testis) gene, and a bromodomain gene (BRD3 or BRD4) or
other, as yet uncharacterized fusion partner genes. In particular,
it is directed to the treatment of these patients with agents that
promote increased histone modification, especially acetylation by
histone deacetylase inhibitors.
BACKGROUND
[0004] NUT Midline Carcinoma
[0005] NUT midline carcinoma, or "NMC," is a rare form of cancer
characterized by a chromosomal rearrangement in which a portion of
the NUT (nuclear protein in testis) gene on chromosome 15 is fused
to a BRD (bromodomain protein) gene or other, as yet unidentified,
gene (French, et al., Cancer Res. 63(2):304-307 (2003); French, et
al., J. Clin. Oncol. 22(20):4135-4139 (2004); French, et al.,
Oncogene 27(15):2237-2242 (Apr. 3, 2007)). NUT fusion genes encode
oncoproteins that maintain cells in an undifferentiated state and
promote their rapid and uncontrolled growth. The frequent
involvement of midline structures in the head, neck, mediastinal,
and other midline structures, suggest that NMCs arise from
primitive neural crest-derived cells. NMCs are very aggressive
clinically, respond poorly to conventional chemotherapy, and are
almost uniformly fatal.
[0006] BRD4 was originally named MCAP (Mitotic
Chromosome-Associated Protein) because it remains bound to
chromatin via its two bromodomains during mitosis. It is thought to
bind in the region of actively transcribed genes before mitosis,
thus providing a kind of cellular memory that ensures re-initiation
of transcription from these sites after mitosis is completed. Two
recent studies provide evidence that this may indeed be the case
(Yang, et al., Mol. Cell Biol. 28(3):967-76 (2008); Mochizuki, et
al., J. Biol. Chem. 283(14):9040-9048 (2008)). The function of BRD3
is less well characterized but, like all proteins in the BRD
family, it contains two acetyl-histone-binding bromodomains and an
extra terminal domain (Wu, et al., J. Biol. Chem.
282(18):13141-13145 (2007); Thorpe, et al., Gene 200(1-2):177-183
(1997)). BRD3 is highly homologous to BRD4 and so its involvement
in NMC is not unexpected. About two thirds of NMCs result from
fusion of NUT to BRD4, and the remaining result from fusion of NUT
to BRD3 or other, as yet uncharacterized, gene (French et al.,
Oncogene 27 (15):2237-2242 (Apr. 3, 2007)).
[0007] In contrast to BRD proteins, NUT lacks known functional
domains, is poorly conserved, and is apparently restricted to
mammals. Although NUT normally shuttles between the nucleus and
cytoplasm, it remains bound to chromatin when fused to BRD4 or BRD3
(French, et al., Oncogene 27(15):2237-2242 (Apr. 3, 2007)). This
suggests that the BRD moiety of the fusion protein serves to tether
NUT to chromatin, thus modifying the function of either or both
proteins in a way that affects transcription. One important
consequence of BRD-NUT expression has been discovered using siRNA
to silence the expression of BRD3- or BRD4-NUT in NMC cell lines.
It was found that withdrawal of the NUT fusion proteins resulted in
irreversible squamous differentiation and arrested growth (French,
et al., Oncogene 27(15):2237-2242 (Apr. 3, 2007)). These findings
suggest that BRD-NUT proteins block differentiation.
[0008] Histone Deacetylase Inhibitors
[0009] During the last few years, it has become increasingly clear
that the acetylation of histones plays a central role in the
structure of chromatin and gene regulation. Acetylation reduces the
positive charge of histones, thereby relaxing the structure of the
nucleosome and facilitating the interaction of transcription
factors with DNA. Removal of the acetyl group restores the positive
charge, thereby causing the nucleosome to contract and become less
accessible to transcription factors (Wade et al., Trends Biochem.
Sci. 22:128 132 (1997); and Wolffe, Science 272:371-372
(1996)).
[0010] Histone deacetylases (HDACs) catalyze the removal of acetyl
groups from histones and appear to play a particularly important
role in regulating gene expression. HDACs are segregated into four
functionally related classes based on sequence homology to
characterized yeast proteins Inhibition of class I HDACs has been
actively pursued as an anticancer strategy due to epigenetic
changes that affect gene expression in cancer cells. At least one
HDAC inhibitor, vorinostat, has been approved by the FDA for use in
certain cancers. Activity has been documented in hematologic
malignancies, in particular, cutaneous T-cell lymphoma (Minucci, et
al., Nature Reviews 6(1):38-51 (2006); Duvic, et al., Blood
109(1):31-39 (2007)). Dose-limiting toxicities for this class of
drug include fatigue, nausea, lethargy and myelosuppression, in
particular thrombocytopenia.
SUMMARY OF THE INVENTION
[0011] The present invention is based upon the discovery that
cancer cells carrying BRD-NUT chromosomal rearrangements respond to
histone deacetylase (HDAC) inhibitors by becoming more
differentiated and slowing their rate of growth. Thus, HDAC
inhibitors should be effective in treating NUT midline carcinoma, a
rare cancer that is characterized by the presence of these
rearrangements.
[0012] In its first aspect, the invention is directed to a method
of treating a patient that has been diagnosed as having a cancer
characterized by cells with a chromosomal translocation involving
NUT (e.g., BRD4-NUT or BRD3-NUT) or by the presence of bromodomain
proteins that exist as translocation partners (or that are fused to
proteins critical to transcription) by administering a
therapeutically effective amount of a compound that promotes
modification, especially increased acetylation, of histones. The
most preferred compounds are histone deacetylase (HDAC) inhibitor
compounds such as SAHA, FK-228, LBH-589, CRA-024781 (also called
PCI-24781), and MS275, but small interfering RNAs may also be used
either alone or in conjunction with HDAC inhibitors. The term
"therapeutically effective amount" means that sufficient compound
is given to a patient to slow or halt the rate of tumor growth or
cause a reduction in tumor volume. A therapeutically effective
amount may also be evidenced by cells assuming a more
differentiated phenotype, which may be manifested paradoxically and
temporarily as an increase in tumor volume. In general, it is
expected that a patient will be administered a daily dose of 1-2000
mg of HDAC inhibitor, preferably a daily dose of 10-1000 mg, and
more preferably 50-600 mg. The cancers containing NUT
rearrangements will typically be carcinomas of the aerodigestive
tract or mediastinum, especially cancers of the trachea; pharynx;
thymus; nasal cavity; thorax; sinuses; or larynx. However, cancers
with NUT rearrangement also occur less frequently in other
locations, such as bladder and bone.
[0013] The invention also encompasses methods of treating a patient
for a solid tumor in which cells from the tumor are first assayed
to determine whether they carry a NUT chromosomal rearrangement. If
this assay indicates that the chromosomal rearrangement is present,
the patient is administered a therapeutically effective amount of
compound that promotes increased acetylation of histones. Although
any assay, including immunohisto-chemistry demonstrating NUT
expression, may be used to determine whether cells carry a NUT
rearrangement, the preferred assay is a fluorescence in situ
hybridization (FISH) assay or conventional cytogenetics. As
discussed above, histone deacetylase (HDAC) inhibitors, e.g., SAHA,
FK-228, LBH-589, CRA-024781, and MS275 are the most preferred
therapeutic compounds but other compounds promoting increased
acetylation may also be used. Preferred dosages and types of
cancers most typically treated are given above.
[0014] In another aspect, the invention encompasses methods of
determining whether cancer cells will respond to a histone
deacetylase inhibitor by performing an assay (preferably a FISH
assay) to determine if the cells have a NUT chromosomal
rearrangement or express NUT or a NUT-fusion protein.
[0015] The invention encompasses multiwell assay plates containing
serial dilutions of at least one, and preferably 5 or more, histone
deacetylase (HDAC) inhibitors (or compounds being tested for NMC
activity), each well having only one species of compound. Examples
of compounds that may be bound to wells include: CRA-024781; APHA;
bortezomib; Apicidin, CI-994; FK228; HC-Toxin; ITF2357; LAQ824;
LBH589; MGCD0103; MS275; Niltubacin; Oxamflatin; PXD101;
Pyroxamide; SAHA; Scriptaid; TSA; Tubacin; Nialamide; PBA; PBHA;
Phenylzine; Tranylcypromine; VPA; and VPHA. Preferred compounds are
CRA-024781, SAHA, FK-228, LBH-589 and MS275. The plates may be used
for testing the responsiveness of NMC cells to treatment by each of
the bound compounds. This may be accomplished by: a) incubating
test NMC cells in the wells of the multiwell assay plate; b)
assaying the test cells in each well to determine proliferation;
histone acetylation; and/or expression of a protein (such as
keratin) characteristic of cells that have become differentiated;
and c) concluding that the NMC cells are responsive to the compound
present in an assay well if the assay of step b) indicates that,
relative to control NMC cells incubated under the same conditions
but in the absence of compound, the test NMC cells exhibit reduced
proliferation, increased histone acetylation and/or increased
expression of a protein that identifies cells that have become
differentiated. In a preferred embodiment, the cells are analyzed
using antibody that recognizes acetylated histones or keratin.
DETAILED DESCRIPTION OF THE INVENTION
[0016] NUT midline carcinoma (NMC) is a rare, highly lethal cancer
that occurs in children and adults of all ages. NMCs occur in the
midline, most commonly in the head, neck, or mediastinum, as poorly
differentiated carcinomas with variable degrees of squamous
differentiation. This tumor is defined by rearrangement of the
"nuclear protein in testis" (NUT) gene on chromosome 15q14. In most
cases, NUT is involved in a balanced translocation with the BRD4
gene on chromosome 19p13.1, an event that creates a BRD4-NUT fusion
gene. Variant rearrangements, some involving the BRD3 gene, occur
in the remaining cases. NMC may be diagnosed by detection of NUT
rearrangement by fluorescence in situ hybridization, karyotype
analysis, or RT-PCR. Due its rarity and lack of characteristic
histologic features, most cases of NMC currently go
unrecognized.
[0017] NMC Defined Molecularly
[0018] NMC is defined herein as any malignant epithelial tumor with
rearrangement of the NUT gene. In approximately 2/3 of cases, NUT
(chromosome 15q14) is fused to BRD4, on chromosome 19p13.1, forming
the BRD4-NUT fusion gene. In the remaining 1/3 of cases, the
partner gene is BRD3 or other uncharacterized gene. We term these
NUT-variant fusion genes. The histologic features of NMC are not
distinctive, and diagnosis is based on detection of the NUT
rearrangement. NUT rearrangements define NMCs, and for this reason
the diagnosis is never in question once rearrangement of NUT has
been demonstrated.
[0019] Diagnosis
[0020] As noted above, normal NUT expression is restricted almost
exclusively to the testis. Thus, positive nuclear
immunohistochemical (IHC) staining for NUT in tissues outside the
testis is indicative of aberrant expression, such as in NMCs, where
both BRD4-NUT and NUT-variants localize to the nucleus. Testing of
rabbit polyclonal NUT antibodies for diagnostic utility using a
panel of five NMCs and twenty-three NUT-unrelated poorly
differentiation carcinomas of the upper aerodigestive tract
suggests a specificity of 95% and a sensitivity of 60%. This may be
somewhat less sensitive than one would like in a diagnostic test
that is envisioned as a screen for the selection of tumors for
confirmatory FISH testing. However, NUT monoclonal antibodies may
permit the development of a more sensitive, IHC-based diagnostic
screening test.
[0021] Assays for Chromosomal Rearrangements
[0022] In order to carry out assays to determine whether a NUT
rearrangement has occurred in a cancer, tumor cells must first be
obtained, e.g., by fine needle aspiration or a tissue biopsy. Any
assay may then be used to determine whether cells are present
having a rearrangement of the type discussed above. For example,
the polymerase chain reaction may be used to amplify sequences in
regions that would indicate that a fusion has occurred (Engleson,
et al. BMC Cancer 6:69 (2006), incorporated herein by reference in
its entirety). The preferred assay is the fluorescence in situ
hybridization (FISH) assay described in French et al., Am. J.
Pathol. 159:1987-1992 (2001) and French et al., Oncogene 27
(15):2237-2242 (Apr. 3, 2007), incorporated herein by reference in
their entirety. This dual color, split-apart assay is performed on
frozen tissue, air-dried cells, methanol-acetic acid preparation of
metaphase-arrested cells, formalin-fixed, paraffin-embedded,
unstained, 4-.mu.m sections of tumor, or formalin-fixed,
paraffin-embedded, unstained disaggregated thick (50 um) sections
of tumor. Probes used for the BRD4 breakpoint on chromosome 19p13.1
break point included telomeric bacterial artificial chromosome
(BAC) clone 87m17 (green) and centromeric yeast artificial
chromosome (YAC) clone 766e7 (red). Presently, telomeric tandem
BACs, RP11-319010 and RP11-681d10, and centromeric tandem BACs,
RP11-207i16 and CTD-3055m5 are used to assay for the BRD4
breakpoint. Probes used for the 15q13 break point (NUT), flanking a
181-kb region, include telomeric BAC clones 1H8 and 64o3 (green)
and centromeric clones 412e10 (recently replaced with 1084a12) and
3d4 (red). Probes used for the BRD3 (chromosome 9q34.2) include
telomeric BAC clone 145e17 (green), and centromeric BAC clone
2243h5 (red).
[0023] HDAC Inhibitors
[0024] Treatment methods described herein include the use of HDAC
inhibitors. These compounds have been very extensively studied in
the treatment of several diseases, including various types of
cancer. As a result, a very large number of inhibitors have been
developed and some are commercially available. Compounds that may
be used in connection with the present invention are described in:
U.S. Pat. Nos. 7,381,825; 7,381,749; 7,375,228; 7,375,137;
7,368,572; 7,345,043; 7,312,247; 7,291,492; 7,288,567; 7,282,608;
RE39,850; 7,271,195; 7,265,154; 7,253,204; 7,250,514; 7,250,504;
7,244,751; 7,214,831; 7,205,304; 7,193,105; 7,169,801; 7,154,002;
7,135,493; 7,091,229; 7,057,057; 6,905,669; 6,897,220; 6,673,587;
6,638,530; 6,541,661; 6,495,719; 20080146623; 20080139547;
20080139535; 20080132503; 20080132459; 20080112889; 20080108601;
20080096920; 20080039509; 20080033015; 20080015216; 20080015190;
20070293530; 20070292351; 20070281934; 20070213330; 20070149495;
20070142393; 20070135438; 20070135431; 20070135424; 20070122507;
20070105808; 20070037869; 20060264415; 20060235231; 20060199829;
20060167103; 20060148743; 20060106049; 20060052599; 20060047123;
20060030554; 20060030543; 20060020131; and 20050288282. All of
these references are hereby incorporated by reference in their
entirety.
[0025] Pharmaceutical Compositions
[0026] The therapeutic compounds described herein may be
incorporated into pharmaceutical compositions in accordance with
methods that are standard in the art (see e.g., Remington's
Pharmaceutical Sciences, Mack Publishing Co., (1990)). Formulations
may be designed for delivery by any of the routes commonly used in
the art.
[0027] Therapeutic compounds may be used in conjunction with any of
the vehicles and excipients commonly employed in pharmaceutical
preparations including water, salt solutions, alcohols, gum arabic,
vegetable oils, benzo-alcohols, polyethylene glycol, gelatin,
carbohydrates such as lactose, amylase, or starch; magnesium
stearate; talc; salycic acid; paraffin; fatty acid esters;
polymers; etc. The pharmaceutical preparations can be sterilized
and, if desired, mixed with auxiliary agents such as: dispersants;
lubricants; preservatives; stabilizers; wetting agents;
emulsifiers; salts for influencing osmotic pressure; buffers;
coloring agents; flavoring agents; and/or aromatic substances.
[0028] Solutions, particularly solutions for injection, can be
prepared using water or physiologically compatible organic solvents
such ethanol, 1,2-propylene glycol; polyethylene glycol;
polygycols; dimethylsulfoxides; fatty alcohols; triglycerides;
partial esters of glycerine; and the like. The preparations can be
made using conventional techniques and may include sterile isotonic
saline, water, 1,3-butanediol, ethanol, 1,2-propylene glycol,
polygycols mixed with water, ringers Ringer's solution etc.
[0029] Dosage Forms and Routes of Administration
[0030] The present invention is compatible with any route of
administration including oral, peroral, internal, rectal, nasal,
lingual, transdermal, intravenous, intraarterial, intramuscular,
intraperitoneal, intracutaneus, and subtaneous routes. Dosage forms
that may be used include tablets, capsules, powders, aerosols,
suppositories, skin patches, parenterals, sustained release
preparations and oral liquids, including suspensions solutions and
emulsions. The most preferred routes for administration are oral,
by injection, or by infusion.
[0031] If desired, compositions, particularly compositions for
injection, may be freeze-dried and lyophilizates reconstituted
before administration. Dosage forms may include compounds promoting
an increase in histone acetylation as the sole active ingredient or
may include other active agents as well. All dosage forms may be
prepared using methods that are standard in the art and that are
taught in reference works such as Remington's Pharmaceutical
Sciences (Osol, A, ed., Mack Publishing Co. (1990)).
Examples
[0032] The results obtained from experiments, and conclusions drawn
based on the results, may be summarized as follows:
[0033] A. Association of BRD-NUT with Decreased Acetylation and
Transcription
[0034] Expression profiling was performed using two NMC cell lines,
TC-797 (Toretsky, et al., Am. J. Clin. Oncol. 26(3):300-306 (2003))
and PER-403 (Kees, et al., Am. J. Ped. Hematol./Oncol.
13(4):459-464 (1991)) treated with control or NUT siRNA.
Twenty-four hours following knockdown of BRD4-NUT in the two NMC
cell lines, prior to the phenotypic features of differentiation,
the number of upregulated genes was found to vastly outnumber the
number of genes that are downregulated, as quantified on
whole-genome expression array chips (Affymetrix HGU-133 plus
2.0).
[0035] Immunoblots of the NMC cell lines TC-797 and PER-403
(Kuzume, et al., Intn'l J. Cancer 50(2):259-264 (1992)) treated
with NUT siRNA or control siRNA revealed a global increase in
acetylated histone H4, H3K18, and H4K8 in response to NUT siRNA.
Consistent with this was a finding that 293T cells containing a
Tet-inducible BRD4-NUT construct showed globally reduced staining
for the same acetyl-histone marks in response to BRD4-NUT
induction.
[0036] Consistent with a role in transcriptional repression,
BRD4-NUT exhibits dominant-negative activity on a BRD4
transcriptional target, an HIV LTR-driven luciferase reporter
gene.
[0037] B. Reversing BRD-NUT-Induced Chromatin Remodeling
[0038] Immunostaining experiments were performed to compare the
spatial distribution of acetylated chromatin with that of BRD4-NUT
in situ. It was found that acetylated chromatin, in the absence of
BRD4-NUT, is diffusely distributed throughout the nucleus. In
contrast, in the presence of BRD4-NUT, acetylation marks become
speckled and co-localize with BRD4-NUT.
[0039] C. HDAC Inhibition in NMC
[0040] Studies aimed at testing the hypothesis of HDAC inhibitor
therapy in NMC have been conducted using trichostatin A (TSA), and
vorinostat (SAHA, Zolinza.RTM.). Both of these compounds are
regarded as non-selective for class I and II deacetylases and bind
HDAC proteins by chelating the active site zinc atom with a
hydroxamic acid feature. NMC cells cultured in vitro were treated
with TSA in dose- and time-ranging studies. Using
immunofluorescence microscopy, it was found that there was an
increase in histone acetylation with increasing dose and time of
exposure. Interestingly, a redistribution of both BRD-NUT and
acetylation marks from nuclear speckles to a diffuse pattern was
also observed. With further drug exposure, a differentiation
phenotype was observed by bright-field microscopy and by
immunohistochemistry for the epithelial differentiation-associated
protein, keratin. Within 24 hours following TSA (25 nM), NMC cell
lines rapidly differentiate, as assessed by brightfield microscopy,
in a manner similar to that seen when BRD-NUT is inhibited with
specific siRNAs. Specifically, TSA caused changes in cellular
morphology and increases in cytoplasmic keratin staining that are
consistent with squamous differentiation.
[0041] These findings suggest that TSA treatment phenocopies direct
interference with BRD-NUT. Consistent with an effect of TSA due to
HDAC inhibition, rather than off-target effects or secondary
toxicities, treatment of five NMC cell lines with pharmacologic
doses of suberoylanilide hydroxamic acid (SAHA), an FDA-approved
HDAC inhibitor with a spectrum of activities similar to that of TSA
(class I and HDAC6 inhibition), also resulted in differentiation
and arrested growth.
[0042] D. Assembly of Chemical Library of HDAC Inhibitors
[0043] In order to explore the response of NMC cells to HDAC
inhibitors, we assembled a library of compounds shown in Table 1.
Compounds were either purchased commercially or chemically
synthesized and plated in serial dilutions (384 wells) with
appropriate numbers of control, and solvent-only wells.
TABLE-US-00001 TABLE 1 Pharma HDAC Inhibitor Library Name Chemotype
APHA Hydroxamic acid Apicidin Ketone CI-994 Hydroxamic acid
CRA-024781 Hydroxamic acid FK228 Thiol HC-Toxin Epoxide ITF2357
Hydroxamic acid LAQ824 Hydroxamic acid LBH589 Hydroxamic acid
MGCD0103 Benzamide MS275 Benzamide Niltubacin Carboxylic acid
Oxamflatin Hydroxamic acid PXD101 Hydroxamic acid Pyroxamide
Hydroxamic acid SAHA Hydroxamic acid Scriptaid Hydroxamic acid TSA
Hydroxamic acid Tubacin Hydroxamic acid Nialamide MAOI PBA
Carboxylic acid PBHA Hydroxamic acid Phenylzine MAOI
Tranylcypromine MAOI VPA Carboxylic acid VPHA Hydroxamic acid
[0044] E. Development of High-Throughput, High-Content Assays
[0045] A cell based approach was developed for identifying potent
and selective HDAC inhibitors. Nuclear acetylation correlates with
inhibition of class I deacetylases such as HDAC1 and HDAC2. An
automated epiflourescent assay was therefore developed which is
measures histone hyperacetylation. Cells were seeded in 384-well
plate format (500 cells/well) and treated with compound. They were
then fixed and stained with: a) Hoechst (nuclei); b) primary
anti-AcHistone polyclonal Ab; c) anti-Keratin monoclonal antibody;
and d) compatible flurophore-conjugated secondary antibodies. After
automated image acquisition, a custom analysis program was applied
that identifies and masks cells based on nuclear intensity
(Hoechst) and then derives quantitative fluorescent data from the
FITC (AcHistone) image. Subsequent secondary masks were generated
using cytosolic intensity.
[0046] F. Adaptation of the Screening Assay (HCS) to NMC
Culture.
[0047] We have performed studies of HDAC inhibitor effects on NMC
cells in culture using an adaptation of the assay described above.
In studies of the effect of SAHA on NMC (TC797) cells in a 384-well
plate format, we have observed increased histone acetylation
qualitatively in images derived from dose-ranging experiments. In a
first attempt at multiplexed detection and quantification of
effects on cell proliferation, histone acetylation, and keratin
protein expression, we have witnessed clear, dose-response activity
of SAHA in the pharmacologically achievable range (C. approximately
2 .mu.M). It was found that SAHA caused a reduction in cell
proliferation and an induction of increased keratin protein content
that correlated with an increase in histone acetylation
(R.sup.2=0.99).
[0048] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by those of skill in the art that the invention may be
practiced within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
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